A two-dimensional analysis method for milk protein, in which the first dimension is preparative isoelectric focusing (IEF) and the second dimension is analytical gel electrophoresis was developed. The analysis of caseins and skim milk showed good separation between $\alpha$s1- and $\alpha$s2-caseins, and two separate bands in $kappa$-casein which are considered to be the genetic variants A and B. This procedure was also useful in the detection of bovine $\alpha$-lactalbumin in the milk of transgenic mice.The major proteins in bovine, caprine, porcine, human, and murine milks were directly compared with a two-dimensional gels which showed characteristic patterns for each milk. Two-dimensional comparison showed that proteins in milk with similar functions were similarly located on the two-dimensional map. Immunological homologies between bovine milk proteins and those of other species were easily examined by this method; ruminant milk proteins showed the greatest similarity, and mouse and ruminant milk proteins showed the least similarity. $\alpha$-Lactalbumin of cow and goat milks resolved as two bands, normal and glycosylated forms of $\alpha$-lactalbumin.Milk fat globule membrane (MFGM) proteins in washed cream were analyzed in two-dimensional gel. Five major MFGM protein bands whose apparent molecular weights of 150, 67, 62.5, 51, and 49 kDa were observed with the separation of band-5 (49 kDa, PAS-7) from band-4 (51kDa, PAS-6) by their different focused fractions in preparative isoelectric focusing.Heat induced effects (72$\sp\circ$C and 87$\sp\circ$C for 2.5 to 60 min) on whole milk proteins were also investigated using this method. $\beta$-Lactoglobulin and other serum proteins interacted readily with MFGM proteins at 87$\sp\circ$C, but only slightly at 72$\sp\circ$C. The interactions observed could not be explained solely by disulfide linkage formation between serum proteins and MFGM proteins. We observed that some MFGM proteins, especially 49 kDa-protein (band-5), were very sensitive to heat treatment. Further characterization of the 49kDa protein demonstrated that binds Concanavalin-A, and its amino acid composition and N-terminal sequence were determined and compared to those reported in the literature.